HTLV III or Human T-cell Leukemia Virus Type III, now commonly referred to as Human Immunodeficiency Virus ("HIV") is recognized to be the causative agent for human immunodeficiency syndrome or AIDS. The chronic nature of AIDS considered as approaching epidemic proportions in the United States and other countries is reflected by a surfeit of studies and efforts to develop diagnostic immunoassays for reliably and consistently detecting viral antigens and antibodies to such antigens in human peripheral blood. In substantial measure, monoclonal antibody technology has been recorded for developing such immunoassays.
HIV belongs to the retrovirus group of viruses. Retroviruses carry a positive-stranded RNA and a special enzyme called reverse transcriptase in its core which is used to convert viral RNA into DNA. This reverses the classical process of cellular transcription in which DNA is converted to RNA.
It is known that the HIV binds to CD4 lymphocytes because the protein on the surface of CD4 lymphocytes serves as a receptor or binding site for HIV [Dalgleish AG et al., Nature 312:763-767 (1985)]. HIV also can bind to and attack other cells, such as, monocytes, tissue macrophages, and cells in the brain, spinal cord and peripheral nerves. The life cycle of HIV calls for the virus entering the host patient through sexual activity or blood transfusion, for instance, and then binding to receptors on monocytes and lymphocytes. The virus penetrates the cell and sheds its envelope or protein coat so as to expose its viral RNA core. The reverse transcriptase converts the viral RNA core to DNA which is integrated into the host cell genome. New viral particles are produced in quantity until the membrane of the host cell is ruptured to release the new viral particles in the human blood system.
Diagnostic tests are commercially available at this time utilizing monoclonal antibodies to determine whether a person having the AIDS disease or has been immunologically exposed to the virus can be identified. Diagnosis of the disease is complicated by the fact that extended periods of incubation are required before symptoms of the disease are expressed. The highly infectious nature of the disease and the fact that its cure presently is not within scientific capability also increases the difficulty of investigating live virus and its adverse affect on the human immune system so that successful diagnostic tests can be developed.
Presently, absolute CD4 count is a generally accepted predictor of clinical HIV disease progression. Current methods for this measurement require multi-step sample preparation and are known to have cumulative errors related to determining both the proportion of CD4 lymphocytes and the absolute lymphocyte count. One method which attempted to solve this problem was presented in a "poster" at the Seventh International Conference on AIDS in Florence, Italy, Jun. 19, 1991. However, the disclosed method was incomplete since it did not teach nor enable one to practice the method.
Similarly, others have attempted to provide different methods and apparatuses to solve the general problem of determining the cell count per unit volume of particles suspended in a fluid containing undesirable particles. One approach is described in U.S. Pat. No. 4,989,978 to Groner. However, this method requires that a predetermined known volume of a suspension of cells and undesirable particles be analyzed so that the volume of the undesirable particles may be subtracted from the original predetermined volume to provide an accurate count of cells per unit volume.
More generally, others have added a plurality of particles to various diluents in methods for testing and calibrating flow cytometry instruments. Although the prior art contains many such disclosures, two such examples are U.S. Pat. No. 4,704,891 to Recktenwald, et al and U.S. Pat. No. 4,331,862 to Ryan. In addition, by adding standard particles to a cell suspension prior to analysis, a "built in" fiducial mark is provided which can be used to interrelate distributions obtained under differing instrument conditions. According to Flow Cytometry and Sorting, John Wiley & Sons Publisher, 1979, this built in technique is taught by Jensen, et al, Multiparameter Flow Cytometry Applied Toward Diagnosis of Cervical Squamous Cell Carcinoma. Proceedings of the Second International Conference on Automation of Cancer Cytology and Cell Image Analysis, May 6-7, 1977. However, these general references fail to address the problem of determination of cell count per specimen volume.